Caccuri A M, Polizio F, Piemonte F, Tagliatesta P, Federici G, Desideri A
Department of Biology, University of Rome Tor Vergata, Italy.
Biochim Biophys Acta. 1992 Aug 21;1122(3):265-8. doi: 10.1016/0167-4838(92)90402-y.
A spin-labelled analogue of glutathione (sl-glutathione) has been used in order to characterize the active site of human placenta glutathione transferase pi. The sl-glutathione shows a competitive inhibition towards glutathione (Ki = 14 microM). Binding of sl-glutathione to the enzyme, followed by electron paramagnetic resonance spectroscopy, gives a Kd of 3 microM and two identical binding sites for dimeric unit. Inhibition of the enzyme, by modification of the Cys-47 residue, completely prevents the binding of sl-glutathione. The same results are obtained by monitoring the binding of glutathione by means of fluorescence spectroscopy. It is concluded that integrity of the thiolate of Cys-47 is necessary to maintain an active conformation of the enzyme able to efficiently bind glutathione into the active site.
为了表征人胎盘谷胱甘肽转移酶π的活性位点，已使用谷胱甘肽的自旋标记类似物（sl-谷胱甘肽）。sl-谷胱甘肽对谷胱甘肽表现出竞争性抑制作用（Ki = 14 μM）。通过电子顺磁共振光谱法跟踪sl-谷胱甘肽与该酶的结合，得到的解离常数Kd为3 μM，并且二聚体单元有两个相同的结合位点。通过修饰半胱氨酸-47残基来抑制该酶，可完全阻止sl-谷胱甘肽的结合。通过荧光光谱法监测谷胱甘肽的结合也得到了相同的结果。得出的结论是，半胱氨酸-47硫醇盐的完整性对于维持能够有效将谷胱甘肽结合到活性位点的酶的活性构象是必要的。
